Abstract

Uterine leiomyosarcoma (ULMS) is a type of uterine sarcoma that accounts for approximately 1% of all uterine malignancies. Existing therapies for this type of cancer are not very effective as evidenced by the high recurrence rate. The rarity of the disease precludes physicians from organizing informative clinical trials and scientists from obtaining sufficient materials to study ULMS on a molecular level. A poor understanding of the biology of the disease, in turn, limits the ability to develop effective treatment strategies. Thus, there is a great need for preclinical models that recapitulate the pathophysiology of the human disease. We generated a mouse model of ULMS in which we showed that BRCA1 can augment tumor progression. Furthermore, we showed that BRCA1 is silenced in a subset of human ULMS, indicating that it may play a role in human tumor development. Currently, there is much excitement about therapies that target the BRCA pathway since early clinical trials with such therapies are showing considerable efficacy in breast and ovarian cancer patients with BRCA-associated tumors. We think that uterine LMS patients with a nonfunctional BRCA pathway may be candidates for therapy that targets cells with DNA repair deficiency.

Uterine Sarcomas

Uterine sarcomas are relatively rare tumors of mesenchymal origin that can be divided into four major histologic subtypes: carcinosarcomas, leiomyosarcomas, endometrial stromal sarcomas, and adenosarcomas. Carcinosarcomas account for 50% of uterine sarcomas, leiomyosarcomas comprise about 30%, and endometrial stromal sarcomas and adenosarcomas each account for about 8% of these tumors.1,2

Despite representing a low fraction of uterine cancers, sarcomas account for a disproportionately high fraction of deaths. A study of 157 patients with various histologic subtypes of uterine sarcoma showed an overall five year survival rate of 40%.3 Of these, ULMS histology predicted the worst prognosis. Even patients with early stage (I & II) disease have been demonstrated a recurrence rate of approximately 70%, with the sites of recurrence often being distant.4

Uterine Leiomyosarcoma Staging

The system used for staging uterine sarcomas is the FIGO (International Federation of Gynecology and Obstetrics) system of staging. This is a surgical staging system, which means that the stage of disease is assigned during the operation upon determination of the extent of disease.

Stage I: The cancer is only found in the main body of the uterus. It has not spread to the cervix, lymph nodes, or distant sites.

Stage IA: The cancer is only in the lining of the uterus (the endometrium).

Stage IB: The cancer has spread from the endometrium into the myometrium (muscular wall of the uterus), growing less than halfway through the myometrium.

Stage IC: The cancer has spread from the endometrium into the myometrium, growing more than halfway through the myometrium. The cancer has not spread beyond the body of the uterus.

Stage II: The cancer has spread from the body of the uterus to the cervix (the lower part of the uterus next to the vagina). The cancer has not spread to lymph nodes or distant sites.

Stage IIA: The cancer involves the body of the uterus and the lining of the cervix.

Stage IIB: The cancer is in the body of the uterus and has grown through the lining of the cervix and into the supporting connective tissue (called the cervical stroma).

Stage III: The cancer has spread outside the uterus but is still only in the pelvic area.

Stage IIIA: The cancer has not spread to lymph nodes or distant sites, but it has spread to: the layer of tissue on the outer surface of the uterus (the serosa), the fallopian tubes or ovaries (adnexa) or the peritoneal fluid (fluid from the inner lining of the pelvis and abdomen).

Stage IIIB: The cancer has spread beyond the uterus to the vagina. It has not spread to lymph nodes or distant sites.

Stage IV: The cancer has spread to the inner surface of the urinary bladder or the rectum (lower part of the large intestine), to lymph nodes in the groin, and/or to distant organs, such as the bones or lungs.

Stage IVA: The cancer has spread to the inner lining of the rectum or urinary bladder (called the mucosa). It may also be in the lymph nodes but has not spread to distant sites.

Stage IVB: The cancer has spread to organs that are not next to the uterus (such as the bones or lungs) or it has spread to distant lymph nodes (such as those in the groin area).

Uterine Leioyomyosarcomas

Figure 1: Histology of human (A) and mouse (B) uterine LMS...

Upon gross examination, most ULMS appear as large, poorly circumscribed masses with a fleshy surface and areas of hemorrhage or necrosis. On a microscopic level (Figure 1A), the final diagnosis is established when any two of the three following criteria are present: A. diffuse moderate to marked cytologic atypia; B. 10 or more mitoses per high power field; C. tumor cell necrosis.2,5,6 Tumor grade and stage appear to be the strongest prognostic variables. Some studies have found that favorable prognostic features may include premenopausal status, low mitotic count, and absence of necrosis.4,7-10

The primary treatment of ULMS is surgery. Total abdominal hysterectomy and bilateral salpingoophorectomy is the standard operation performed for resection of this type of tumor. Pelvic and/or para-aortic lymphadenectomy is not indicated for uterine leiomyosarcoma, in which retroperitoneal involvement is rare unless macroscopic extra-uterine disease is present.4,11,12 Chemotherapy treatment practices are highly variable.8,13,14 This is largely due to a paucity of prospective randomized clinical trials examining the outcome in individuals with ULMS due to the intrinsic rarity of this disease.

A search for treatment modalities leading to improved survival and decreased recurrence of ULMS has yielded extremely limited results. Although pelvic radiation therapy has been shown to decrease the local recurrence rate, it did not significantly improve chances of survival in individuals with ULMS.4,15 Similarly, until recently no adjuvant regimen of chemotherapy has been shown to improve survival. A notable exception to this track record is a phase II study of adjuvant treatment with gemcitabene and docetaxel in patients with completely resected stage I-IV, high grade ULMS that resulted in improved two-year progression free survival.16

The etiology associated with the carcinogenesis of ULMS is poorly understood. Frequently observed mutations and overexpression of p53 in ULMS suggest that the loss of p53 function may play a critical role in the development of this cancer.17-19 Other common molecular features of ULMS include increased expression of p1618,20-22 and the presence of nuclear b-catenin.23,24

A Mouse Model Reveals a Possible Role for BRCA1 in ULMS

We generated a genetically engineered mouse tumor model25 in which mice develop uterine tumors that resemble the histology of human ULMS, including spindle shaped cells with hyperchromatic nuclei, prominent nucleoli, abundant mitoses and marked cytological atypia (Figure 1B). Similar to human ULMS, the mouse tumors exhibited positivity for vimentin, overexpression of p16, and the presence of nuclear b-catenin.25

Genetically Engineered Mouse Tumor Models

Mice designed with specific genetic modifications that alter expression of oncogenes or tumor suppressor genes. The mice typically develop tumors that recapitulate some aspects of human disease and offer an opportunity to evaluate the efficacy of therapies targeted towards the genes of interest.

Figure 2: Schematic presentation of uterine LMS development in mice...

In the mouse model of ULMS, the p53 and/or BRCA1 tumor suppressor genes were conditionally inactivated using a tissue-specific Cre-Lox system where Cre is driven by the female reproductive system-specific Amhr2 promoter. We demonstrated that conditional deletion of p53 in the female reproductive tract can result in ULMS development and that concurrent inactivation of p53 and BRCA1 further augments tumor progression (Figure 2).

The p53 tumor suppressor gene is known to play a role in human ULMS development,17,18,22,26 but the role of the BRCA1 tumor suppressor gene has not been explored. Women with germline BRCA1 mutations are predisposed to breast and ovarian cancers but not ULMS, indicating that genomic alterations of BRCA1 are unlikely to play a role in the development of this disease. However, genetic or epigenetic somatic inactivation of BRCA1 may contribute to ULMS progression. Consistent with this hypothesis, immunohistochemical detection of the BRCA1 protein revealed that its expression is lost in more than a quarter of human ULMS.25 This observation suggests that BRCA1 silencing may play a role in the development or progression of human ULMS. In addition to germline mutations, the loss of BRCA function may result from somatic mutations, loss of heterozygosity (LOH), and promoter hypermethylation. We are currently exploring whether one or more of these mechanisms are responsible for BRCA1 silencing in those ULMS samples without detectable BRCA1 protein expression.

Tissue-specific Cre-Lox Conditional Knockout Mouse Models

Genetically engineered mice in which a gene of interest is flanked by two specific inverted repeat DNA elements, such as loxP sites. A bacterial enzyme, Cre recombinase, catalyzes a recombination between the loxP sites resulting in a deletion of the gene of interest. Cre recombinase can be expressed from a tissue-specific promoter to inactivate gene expression in that specific tissue.

The Role of BRCA1 in Cancer

Germline mutations in the BRCA1 tumor suppressor genes are associated with increased risk and earlier onset of breast and ovarian cancers.30 The specific mechanism of BRCA1-associated tumorigenesis is not well understood, although a lot has been learned about this gene since its discovery in 1994.30,31 The BRCA1 gene encodes a 220-kd nuclear phosphoprotein that plays an integral role in responding to cellular stress, localizing to sites of damaged DNA and activating specific repair processes.32 BRCA1 is known to interact with numerous genes and proteins collectively known as the BRCA pathway; defects in this pathway are believed to be a driving force in cancer progression.33-35

PARP-1 Targeted Therapy in a Mouse Model of ULMS

Our mouse model of BRCA1-wild type and BRCA1-deficient ULMS (Figure 2) provides a unique experimental system for the evaluation of therapies that target the BRCA1 pathway. We will test whether poly(ADP-Ribose) Polymerase-1 (PARP-1) inhibitors are effective in reducing tumor burden in BRCA1-wild type and BRCA1-deficient mice. We anticipate that PARP-1 inhibitors will be more effective in reducing tumor burden in BRCA1-deficient ULMS than in BRCA1-wild type ULMS. This assumption is based on existing data from breast and ovarian cancer studies where it has been demonstrated that BRCA-deficient cells are up to 1,000 times more sensitive to PARP-1 inhibitors than cells that contain a functional BRCA pathway.27,28 Consequently, PARP-1 inhibitors are highly selective in targeting tumor cells based on their lack of BRCA activity, while normal tissues surrounding the tumor are not affected by PARP-1 inhibition. Since PARP-1 inhibitors only target tumor cells, this therapy is presumed to be relatively safe and with minimal side-effects.29 We expect that our research will provide justification for the development of PARP-1 inhibitor clinical trials for ULMS patients and, ultimately, lead to a safe and effective treatment option for women with ULMS.

The involvement of BRCA1 and BRCA2 genes in DNA repair has prompted researchers and clinicians to investigate whether tumors with mutations in these genes would be more susceptible to treatments that generate double-strand DNA breaks, such as radiation and platinum agents.36,37 One novel treatment that takes advantage of the inability of BRCA-deficient cells to repair DNA damage is the inhibitory effect of PARP-1. PARP-1 is an enzyme that is responsible for repairing single-strand DNA breaks.38,39 Its inhibition results in multiple single-strand breaks along DNA. Such single-strand breaks can progress into double-strand breaks through the collapse of replication forks. The preferred mechanism for repair of double strand-breaks is homologous recombination which relies on the activity of the BRCA genes. Thus, BRCA-deficient cells are more sensitive to single-strand breaks than normal cells. Consequently, PARP-1 inhibitors are specific in targeting tumor cells while normal tissues are spared.

Schematic presentation of uterine LMS development in mice with conditional inactivation of BRCA1 and/or p53 in reproductive organs. The percentage of mice that succumb to tumor development within 13 months indicates that conditional deletion of BRCA1 augments tumor formation in mice with conditional deletion of p53.

Grant Funding

The Liddy Shriver Sarcoma Initiative awarded this $25,000 grant in August 2009. The study was made possible, in part, by generous donations made in memory Suzanne Kurtz and Teal Harris, who lost their lives to leiomyosarcoma; and by generous donations made by the family and friends of Jim Hauser, who is fighting leiomyosarcoma.